Peptide Purification - (Apr/06/2004 )
I have a peptide, which is found in three forms. Unphosphorylated,
singly phosphorylated and doubly phosphorylated. I observed this data
by MALDI-TOF. Actaully this peptide was just send me, and my professor
asked me to look at the mass data, and this is what came from this
Peptide ,which is about 20 aa long, has binding affinity to my
protein, and I want to look at the binding affinity of
unphosphorylated, singly phosphorylated and doubly phosphorylated
peptide to my protein by ITC.
What I need is the way of seperating (purifiying from each other)
these peptides from each other. Size-exclusion and ion-exchange did
not help at all. I would be pleased if you can adress a method on this
What is the site of phosphorylation on the peptide? You might need a careful microfractionation of your peptide mix and we recommend a immunoaffinity column having agarose cross-linked to the particular phosphoaminoacid antibody to catch your isoforms. I think Sigma has a collection of these agarose gels.
Why do not use isofocusing or native electroforesis followed by exclusion of peptides from gel?
I would suggest HPLC as the method of choice - with a prep colummn you should be able to purify enough for ITC